Protease-catalyzed small peptide synthesis in organic media

ELSEVIER

Protease-catalyzed small peptide synthesis in organic media Xue-zhong Zhang, Xu Wang, Songming Chen, Xueqi Fu, Xiaoxia Wu, and Changhao Li State Key Laboratory

of Enzyme Engineering,

Jilin University, Changchun,

China

Four kinds of supports were used to prepare immobilized papain using different methods: simple absorption on Celite, ionic absorption on CM-cellulose and QAE-Sephadex, and covalent cross-linking on egg white protein. The direct effects of the support on the catalytic properties of the enzyme were examined by studying the dependence of water content, pH, ionic strength, and reaction temperature on the yields of a model dipeptide Boc-Phe-ValOMe in ethyl acetate under thermodynamic control. It was found that the supports with low hydrophilicity demonstrated much higher catalytic activity by the enzyme than those with high hydrophilicity. Egg white protein was the best support with a good yield (94.5%) of the dipeptide. Papain and a-chymotrypsin immobilized on egg white protein were used to synthesize a number of dipeptides with Boc-Phe-XOMe showing different substrate specificity. Alcalase was used in the synthesis of some dipeptides containing hydrophilic amino acids and n-amino acids with reasonable yields. 0 1996 by Elsevier Science Inc. Keywords:

Small peptide synthesis;

immobilization

supports;

Introduction Enzymatic catalysis in nonaqueous media has attracted a great deal of attention in recent years.‘** Hydrolysis of the peptide bond catalyzed by protease can be reversed for peptide bond formation under certain conditions. A few of the proteases have been successfully applied to the synthesis of a number of small peptides of pharmaceutical and nutritional interest such as enkephalin,374 aspartame precursor,5 and some nutritional dipeptides and tripeptides.6*7 Some small peptides can be continuously synthesized at the commercial scale by using efficient enzyme reactors*-“; however, because of the characteristics of enzymatic catalysis in nonaqueous media, a great deal of effort has been made to improve methodology mainly involving enzyme stabilization,’ ’ the search for new enzymes,‘* the solubility of hydrophilic amino acids in apolar organic solvents,” the use of D-amino acids for some useful peptides13*14 with the aim increasing peptide yield. Papain has proven to be a versatile protease for synthesis

Address reprint requests to Dr. Xue-zhong Zhang, State Key Laboratory of Enzyme Engineering, Jilin University, Changchun 130023, Peoples Rep. of China Received 28 September 1995; revised 2 January 1996; accepted 10 January 1996

Enzyme and Microbial Technology 19:538-544, 1996 6 1996 bv Elsevier Science Inc. 655 Aver&e of the Americas, New York, NY 10010

organic

solvents

of a variety of peptides due to its broad substrate specificity. l&l6 In order to study the direct influence of the support on the catalytic properties of this enzyme, in this study we used four kinds of supports to prepare the immobilized papain by different immobilization methods: simple absorption (Celite), ionic absorption (CM-cellulose and QAESephadex) and covalent cross-linking (egg white protein). The direct effects of the supports on the catalytic properties of the enzyme were examined by studying the dependence of water content, pH, ionic strength, and reaction temperature on the yields of a model dipeptide Boc-Phe-ValOMe in ethyl acetate, a water-immiscible organic solvent under thermodynamic control. In addition, the enzymatic substrate specificity for synthesis of several hydrophobic dipeptides in the form of Boc-Phe-XOMe were examined using papain and o-chymotrypsin immobilized on egg white support. Although it was successful to synthesize hydrophobic amino acid-containing dipeptides or tripeptides in high yields, relative few attempts have been made to synthesize hydrophilic amino acid-containing small peptides due to the very low solubility of the hydro hilic amino acids in apolar solvents. Recently, Chen et al. P4*17 reported that an industrial protease, alcalase, was very useful in peptide synthesis not only due to its good stability and high activity in polar alcoholic solvents but also because of its capacity to accept both D- and L-amino acid residues as nucleophiles. In this study, we used alcalase to synthesize hydrophilic dipeptides

0141-0229/96/$15.00 PII SO141-0229(96)00057-9

Protease-catalyzed and n-amino acid-containing dipeptides in ethanol thermodynamic control with reasonable yields.

under

Materials and methods Materials Papain was purchased from Merck (Darmstadt, Germany). a-Chymotrypsin was from Serva Feinbiochemica (Heidelberg, NY). Alcalase was from Novo (Denmark). Casein was from a local supplier. Immobilization supports were QAE-Sephadex A-25 (an anion ion-exchange resin) from Pharmacia (Sweden). Trisacryl M CM (CM-Cellulose, a cation ion-exchange resin) was from IBF LKB Co. Celite was obtained from a local supplier. The egg white protein support was prepared in this laboratory. Protected amino acids were Boc-amino acids, Cbz-amino acids. amino acid methyl ester and Gly-Gly dipeptide which were purchased from Sigma Chemical (St. Louis, MO). Gly-GlyOEt was prepared from the dipeptide Gly-Gly in this laboratory.‘8 Solvents were ethyl acetate, methanol, ethanol, propanol, t-butanol, dioxane, dimethylformamide (DMF), and triethylamine. These and other reagents were analytical grade. Acetonitrile was HPLC grade.

Preparation of the immobilized papain on the different supports Immobilized papain on CM-Cellulose (IPCC) or QAE-Sephadex A-25 (IPQS) was prepared under optimum conditions for recovery of the enzymatic activity. The immobilization coefficient by these methods was reported previously.” For immobilized papain on celite (IPC), 200 mg papain was dissolved in 10 ml of 0.3 M phosphate buffer pH 6.2. Celite (1 .O g) was added to the enzyme solution followed by the addition of 1.O ml of 0.005 M Cys and 0.002 M EDTA. The mixture was shaken at 4°C overnight and then lyophilized. For immobilized papain on egg white protein (IPEW), the egg white support was prepared by the method of Kamata et uZ.*~Egg white powder (5.0 g) was suspended in 50 ml of 50% glucose buffer pH 5.0 and incubated for glucosylation at 80°C for 15 h. It was then washed with 1 1 of distilled water, dried in vucuo, and lyophilized. The resulting powder of egg white support was ground to give a fine powder (30-50 mesh). Egg white support (2 g) was added to 50 ml of papain solution (50 mg ml-‘). The mixture was shaken at 4°C for 72 h. Before lyophilization, the immobilized enzyme was washed with the buffer to remove free enzyme.

Assays for activity of papain and protein determination Assays for free and immobilized papain were carried out by the method of Amon.” Casein (2%) was used as a substrate. The reaction temperature and time were 37°C and 10 min, respectively. Folin’s reagent was used for coloration with OD measurement at 650 nm. Protein concentration was determined by the method of Lowry et a1.22

Determination of water content in organic solvents The water content in organic solvents was measured by the KarlFischer method.

peptide synthesis:

X.-Z. Zhang

et al.

Enzymatic peptide synthesis In experiments studying the effects of different factors (water content, pH, ionic strength, and temperature) on the yield of the dipeptide Boc-Phe-ValOMe for the different supports, the choice of fixed parameters was based on previous conditional experiments. The amount of change for a parameter was chosen on the basis of a papain-catalyzed reaction in aqueous solution. For a typical reaction, a certain amount of immobilized enzyme (corresponding to 0.1 g papain powder) was added to 20 ml of the substrates (60 mM Boc-Phe and 120 mM ValOMe) in ethyl acetate containing 0.54 ml of 0.1 M phosphate buffer pH 6.29 (2.7% water content). The reaction mixture was incubated with shaking at 37°C for certain time. Finally, the reaction solution was evaporated under reduced pressure to dryness. The residue was dissolved in 0.5 ml of a mixture of acetonitrile and water (1: 1) for HPLC analysis. The specific experimental condition for experiments determining the effects of the various parameters on the yield of the dipeptide Boc-Phe-ValOMe can be found in the legends of the Figures. For preparation of Gly-GlyOEt, 10 g Gly-Gly was suspended in 100 ml of ethanol. Anhydrous HCL gas was passed through the suspension with stirring. After Gly-Gly was dissolved completely. stirring was continued for 1 h. Ethanol was removed under reduced pressure. The residue was dissolved with ethanol again. The same procedure was repeated several times to remove excess hydrogen chloride. Finally, Gly-GlyOEt was precipitated out from the solution containing ether and water (2: 1). For dipeptide synthesis catalyzed by alcalase, first the water in the alcalase solution was removed with anhydrous ethanol by the method of Chen et al.” Alcalase (0.1 g) was added to 20 ml ethanol containing a certain amount of phosphate buffer with optimum ionic strength and pH, 50 mM substrate, and 75 mM nucleophile to start the reaction. The reaction mixture was incubated with shaking at 50°C for a certain time. A sample was taken for HPLC analysis.

Analysis of peptide synthesis The reaction progress of peptide synthesis was monitored by TLC analysis performed on silica gel GF254 precoated plates. The solvent system consisted of water, trichloromethane, and methanol (1:8:3 v:v:v). Quantification of the substrates or the peptide products was carried out by high-performance liquid chromatography (HPLC) with a Resolve C,, column (Waters, Milford, MA). The eluant was prepared by mixing A (0.1 M ammonium formate adjusted to pH 6.0 with 10% acetic acid and B (acetonitrile) in the ratios of A:B = 100:55, A:B = 40:60, and A:B = 25175. Calibration curves were constmcted from the areas of peaks of the purified products at 254 nm. Peptide products were identified by analysis of amino acid components; they were dissolved in 6 M HCI and hydrolyzed at 110°C under a vacuum for 24 h. The amino acid analysis was performed on a Hitachi amino acid automatic analyzer (model 835-50).

Results and discussion The synthesis of Boc-Phe-ValOMe catalyzed by the immobilized papain on the various supports Boc-Phe-ValOMe was chosen as the model for hydrophobic dipeptides and the thermodynamically controlled synthesis of the dipeptide catalyzed by the immobilized papain on the various supports in the water-immiscible organic solvent ethyl acetate. After comparing the solubilities of a

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Papers variety of hydrophobic protective amino acids including Boc-Phe and ValOMe in the different organic solvents (methanol, ethanol, propanol, t-butanol, acetonitrile, acetone, benzene, DMF, ethyl acetate, dioxane, etc.), we found that ethyl acetate was a good candidate for the organic solvent used in the synthesis of hydrophobic dipeptides. The reactions for dipeptide synthesis catalyzed by free papain (enzyme powder) and the four kinds of immobilized papain are shown in Figure 1. The synthesis reactions catalyzed by all five forms of papain essentially reached constant after 72 h with the different maximum yields. A number of factors affecting the shift of the chemical equilibrium of the reaction to peptide bond formation were examined.

Effect of water content on the dipeptide synthesis. The water content in the reaction system (the volume of the buffer added to the total volume of 20 ml) is the most important factor affecting the chemical equilibrium of the reaction. Although the volume of the buffer added to the reaction system was the same for the different supports, the effect of water content on the yield of Boc-Phe-ValOMe was observed (Figure 2). Obviously, this relates to the properties of the supports. The enzyme and water combination is essential for enzyme catalysis. The influence of the support on enzyme catalysis is partially due to the competition for water between the support and the enzyme.23 The capacity of absorbing water for the different supports can be very different. CM-cellulose and egg white protein absorb little water while Celite and enzyme powder absorb more water. It can be seen that egg white-immobilized papain (IPEW)

gave the best results with the highest yield (94.5%) at a lower water content (2.7%). The better support is Celite. In contrast, the enzyme powder gave a maximum yield (only 51%) of the dipeptide product at a much higher water content (5%). The higher water content in the reaction system is not favorable thermodynamically for peptide bond formation.

Effect of reaction system pH on dipeptide synthesis. Although the four kinds of immobilized papain were prepared under the optimum pH in aqueous solution, one can expect that the pH dependence of the reaction system for each support used in this study on the yield of the dipeptide might be different (pH of the reaction system refers to the pH of the buffer added). Figure 3 shows that the maximum yields were reached at about pH 7.0 for the immobilized papain, IPEW, IPC, and the enzyme powder. Egg white is the best support with the highest dipeptide yield of 92.5% compared to the yield (51%) for enzyme powder. These two supports are uncharged. For the charged supports (QAE-Sephadex and CM-cellulose), however, the situation was very different. The optimum pH of the reaction system is on the alkaline side for QAE-Sephadex (an anion exchange resin) and on the acidic side for CM-cellulose (a cation exchange resin). It seems that the charge on the support has an effect on the microenvironment of the enzyme thereby resulting in a change in the optimum pH of the reaction. Effect of ionic strength of reaction system on dipeptide synthesis. Figure 4 shows the dependence of the ionic

Figure 1 The reaction courses of Boc-PheValOMe synthesis catalyzed by free papain and the four kinds of immobilized papain. Reaction conditions: the amount of immobilized papain on the different supports corresponds to 0.1 g papain powder; 0.1 M phosphate buffer pH 6.29; 60 mM Boc-Phe, 120 mM ValOMe; water content, 2.7%; reaction temperature, 37°C; and reaction time, 72 h

540

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Protease-catalyzed

peptide

synthesis:

X.-Z. Zhang

et al.

loo -

80-

Q-

IY

---L.--r-r

0.0

2.0

4.0 water

6.0

content

10.0

(96)

I

I-I

5.0

6.0

6.0

7.0

8.0

Figure 2 The effect of the reaction system water content on Boc-Phe-ValOMe synthesis catalyzed by free papain and the four kinds of immobilized papain. Except for the change in water content (the volume of 0.1 M phosphate buffer pH 6.29 added to the total volume of 20 ml), the reaction conditions were the same as in Figure I

\,

-

9.0

PH Enzyme Microb. Technol.,

Figure 3 The effect of the reaction system pH on Boc-Phe-ValOMe synthesis catalyzed by free papain and the four kinds of immobilized papain. Except for the change in pH of the reaction system due to the pH of the buffer used, the reaction conditions were same as in Figure 1

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ionicstrength (M) strength of the reaction system for the different immobilized papains and the enzyme powder on the dipeptide yield. Apart from the enzyme powder, the immobilized papains on uncharged supports need lower ionic strength than that on the charged supports for the maximum dipeptide yield. An increase in ionic strength on the right site of the peaks sharply decreased the dipeptide yield. At an ionic strength of 0.012 M phosphate buffer, the dipeptide yield for IPEW reached a maximum (92%); this was the best support compared to the enzyme powder and the other immobilized papain. Effect of reaction temperature on the dipeptide synthesis.Figure 5 shows the dependence of the reaction temperature on the dipeptide yield for the four kinds of immobilized papain and the enzyme powder. It can be seen that at approximately 40°C all reaction systems reached maximum dipeptide yields. Most of the immobilized papain exhibited much better stabilization against heat denaturation, especially IPEW with the 92% dipeptide yield at temperatures between 40-50°C compared to the enzyme powder. Under the same experimental conditions (0.1 g enzyme powder and equivalent amounts of enzyme immobilized on the different supports; water content, 5%; pH, 6.29; ionic strength, 0.2 M; reaction temperature, 375°C; Boc-Phe, 50 mu; ValOMe, 75 mu; reaction time, 71 h), the dipeptide yields were 51.2% (enzyme powder), 52.5% (IPCC), 67.8% (IPQS), 86.7% (IPC), and 89.4% (IPEW). Compared to the enzyme powder, the immobilized enzymes usually gave better dipeptide yields mainly due to their stability against denaturation by organic solvent and 542

Enzyme Microb. Technol.,

Figure 4 The effect of reaction system ionic strength on Boc-Phe-ValOMe synthesis catalyzed by free papain and four kinds of immobi,:___I -___. In. Except for the change in ionic llLt3” papa of strength dlependent on the concentration phosphate duffer, the reaction conditions were same as in Figure 7

heat; however, the obvious difference in catalytic efficiency between the different immobilized papain has been observed in this study. The properties of the supports and the immobilization methods greatly influenced the catalytic efficiency of the enzyme. Egg white has significant merits over the other supports because of its high stability from covalent cross-linking, little water absorption, and easy suspension in the reaction system which favored the attachment between the substrates and the immobilized enzyme.

The synthesis of hydrophobic dipeptides (Boc-Phe-XOMe) and a tripeptide (Cbz-Tyr-Gly-GlyOEt) catalyzed by immobilized papain on egg white In this study, a few of the hydrophobic dipeptides (BocPhe-XOMe) in which X represents various nucleophiles were synthesized by using IPEW. The sequence Phe-X has been frequently found in several biologically active peptides. Immobilized papain (0.1 g) was added to 20 ml of ethyl acetate containing 50 mu Boc-Phe, 75 mM nucleophile and a certain amount of 0.2 M phosphate buffer pH 6.29. The reaction mixture was incubated at 47.5”C for 72 h. As shown in Table I, the yields for the first four dipeptides (Boc-Phe-ValOMe, Boc-Phe-LeuOMe, Boc-PheMetOMe, and Boc-Phe-AlaOMe) are much higher than those for the last three dipeptides (Boc-Phe-PheOMe, BocPhe-TyrOMe, and Boc-Phe-TrpOMe) containing aromatic amino acids as the donor of the amino group. This result is

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Protease-catalyzed

1w

not surprising because aromatic amino acids do not act as good donors of an amino group to papain. Considering the substrate specificity of o-chymotrypsin in the hydrolysis reaction of the peptide bond and the requirement for an aromatic amino acid residue at the end of a carboxyl group, o-chymotrypsin immobilized on egg white was first chosen to synthesize the dipeptides BocPhe-PheOMe and Boc-Phe-TrpOMe. Immobilized enzyme (0.01 g) was added to 20 ml of ethyl acetate containing 50 mu Boc-Phe, 75 mM nucleophile and a certain amount of 0.1 M phosphate buffer pH 8.2 which makes the water content 2.5%. The reaction system was incubated at 25°C for 72 h. Yields for the dipeptides were 89.5% and 84.5%, respectively. We also used IPEW to synthesize a precursor tripeptide

Table 1 Synthesis of hydrophobic amino acid-containing dipeptide (Boc-Phe-XOMe) in ethyl acetate catalyzed by immobilized papain on egg white protein

Acyl donor Boc-Phe Boc-Phe Boc-Phe Boc-Phe Boc-Phe Boc-Phe Boc-Phe

Nucelophile ValOMe LeuOMe MetOMe AlaOMe PheOMe TyroOMe TrpOMe

Product Boc-Phe-ValOMe Boc-Phe-LeuOMe Boc-Phe-MetOMe Boc-Phe-AlaOMe Boc-Phe-PheOMe Boc-Phe-TyrOMe Boc-Phe-TrpOMe

Water content (%)

Yield (%)

2.7 1.5 1.5 2.2 1.2 3.5 2.3

94.5 92.3 83.5 76.8 42.7 44.5 34.5

peptide synthesis: X.-Z

Zhang et al.

Figure 5 The effect of reactilon temperature on Boc-Phe-ValOMe synthesis catalyzed by free papain and immobilized papain. Except for the change in reaction temperature, the reaction conditions were same as in Figure 1

of enkephalin, Cbz-Tyr-Gly-GlyOEt, which contains the polar amino acid (Gly) in ethyl acetate. The yield was 54.4%. Generally speaking, for a given peptide to be synthesized, it is necessary to choose one or more proteinases on substrate specificity and examine the stability and catalytic efficiency of each in the organic solvents used (i.e., immobilization and effectiveness of support). At the same time it is also necessary to optimize each synthesis step (the main factors influencing chemical equilibrium). In addition, the solubility of substrates, especially hydrophilic amino acids, in apolar organic solvents is another very important factor which must be considered for hydrophilic peptide synthesis. So far, this problem has not been solved well technologically, although some approaches have been reported.‘0,24

Table 2 c-amino calase

Synthesis of hydrophilic amino aaid-containing and acid-containing dipeptides in ethanol catalyzed by al-

Acyl donor

Nucleophile

Boc-Gly Boc-Gly Boc-Phe Boc-His Boc-Phe Boc-Phe Boc-His Cbz-o-Phe

AspOMe GlyOMe LysOMe TrpOMe o-TrpOMe o-PheOMe o-TrpOMe LysOMe

Enzyme Microb. Technol.,

Yield (%I

Product

52.1 68.9 46.7 44.4 59.6 56.7 48.5 52.7

Boc-Gly-AspOMe Boc-Gly-GIyOMe Boc-Phe-LysOMe Boc-His-TrpOMe Boc-Phe-o-TrpOMe Boc-Phe-o-PheOMe Boc-His-o-TrpOMe Cbz-o-Phe-LysOMe

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Papers

The synthesis of dipeptides containing polar amino acid substrates or o-amino acid substrates catalyzed by alcalase

and Matsuno. R. Synthesis of peptides consisting of essential amino acids by a reactor system using three proteinases and an organic solvent. Agric. BioL Chem. 1990. 54, 3331-3333 Monter, B., Herzog, B., Stehle, P., and Ftirst. P. Kinetically controlled synthesis of dipeptides using ficin as biocatalyst. Biotechd Appl. Biochem. 1991. 14, 183-191 Kimura. Y.. Yoshida, T., Muraya, K., Nakanishi, K., and Matsuno, R. Continuous synthesis of a tripeptide by successive condensation and transesterification catalyzed by two immobilized proteinases in organic solvent. Agric. Biol. Chem. 1990. 54, 1433-1440 Herrmann, G., Schwarz, A., Wandrey, C., Kula, M.-R., Knaup, G., Drauz, K. H., and Bemdt, H. Scale-up of enzymatic peptide synthesis in an enzyme membrane reactor. Biotechnol. Appl. Biochem. 1991,13,346-353 Serralheiro, M. L. M., Prazeres, D. M. F., and Cabral, J. M. S. Dipeptide synthesis and separation in a reversed micellar membrane reactor. Enzyme Microb. Technol. 1994, 16, 10641073 Aldercreutz, P. and Mattiasson, B. Aspects of biocatalyst stability in organic solvents. Biocufalysis 1987, 1, 99-108 Pauchon, V., Besson. C., Saulnier, J., and Wallach, A. Peptide synthesis catalyzed by Pseudomonas aeruginosa elastase. Biotechnol. Appl. Biochem. 1993. 17, 217-221 Chen, S-T., Tu, C.-C., and Wang, K.-T. Selective incorporation of n-amino acid esters into peptides catalyzed by alcalase in t-butanol. Bioorg. Med. Chem. Lett. 1993, 314, 539-542 Chen, S.-T., Chen, S.-Y.. Chen, H.-J., Huang, H.-C., and Wang, K.-T. Probing the S-l ’ subsite selectivity of an industrial alkaline protease in anhydrous t-butanol. Bioorg. Med. Chem. Lett. 1993,3, 727-133 Stehle, P.. Bahsitta, H.-P., Monter, B., and Fiirst. P. Papaincatalyzed synthesis of dipeptides: A novel approach using free amino acids as nucleophiles. Enzyme Microb. Technol. 1990, 12, 56-60 Stevenson. D. E. and Storer, A. C. Papain in organic solvents: Determination of conditions suitable for biocatalysis and the effect on substrate specificity and inhibition. Biotechnol. Bioeng. 1991, 37, 519-527 Chen, S.-T., Chen, S. Y., and Wang, K. T. Kinetically controlled peptide bond formation in anhydrous alcohol catalyzed by the industrial protease alcalase. J. Org. Chem. 1992, 57, 6960-6965 Huang, W.-D. and Chen, C.-Q. Polypeptide Synthesis. Academic Press. Beijing, 1985, 81 Zhang, X.-Z., Chen, S., Wang, X., Wu, X., Huang, Z., and Lu, B. A study of dipeptide synthesis catalyzed by protease in organic solvent. In: Ann. N. Y. Acad. Sci. Vol. 750 (Legoy, M.-D. and Thomas, D., Ed%). New York Academy of Sciences Press, New York, 1995, 24-29 Kamata, Y.. Kurota, A., and Yamauchi, F. Enzyme immobilization on glycosylated edible proteins. Agric. Biol. Chem. 1990,54,30493050 Anton, B. Papain. In: Methods in Enzymology XIX (Perimann, G. E. and Lorand, L., Eds.). Academic Press, New York, 1970, p. 226244 Lowry, 0. H., Rosebrough, N. J., Farr, A. L., and Randall, R. J. Protein measurement with Folin phenol reagent. J. Biol. Chem. 195 1, 193, 265-275 Adlercreutz, P. On the importance of the support material for enzymatic synthesis in organic media. Eur. J. Biochem. 1991, 199, 609-614 Kimura, Y.. Tari, Y., Adachi, S., and Matsuno, R. Enzymatic synthesis of peptide with hydrophilic amino acids in water-miscible organic solvent. In: Ann. N. Y. Acad. Sci. Vol. 672 (Clark. D. S. and Estell, D. A., Eds.). New York Academy of Sciences Press. New York, 1992, 458461

7.

Chen et a1.‘3,‘4,‘7 successfully used alcalase to catalyze small peptide synthesis controlled kinetically in anhydrous ethanol and t-butanol; the process gave high yields. In this study, thermodynamically controlled synthesis of dipeptide containing a polar amino acid or txunino acid residue catalyzed by alcalase was performed.

8.

9.

The synthesis of hydrophilic dipeptides. Ethanol

is a strong polar organic solvent. Polar or hydrophilic amino acid substrates with little or low water content are easy soluble in this reagent. The thermodynamically controlled synthesis for four hydrophilic dipeptides were conducted with reasonable yields (Table 2). Compared to the synthesis of the hydrophobic dipeptides (Table I), the yields of hydrophilic dipeptides are much lower. Although there is no problem with the solubility of amino acid substrates in these syntheses, other factor(s) that affect the dipeptide yield such as the catalytic efficiency and substrate specificity of alcalase for the particular target products, might play an important role.

10.

11. 12.

13.

14.

15.

Synthesis of D-amino acid-containing

dipeptide. It was

reported that the S-l subsite of alcalase could accept both D- and L-amino acid residues as nucleophiles.i4 Four kinds of D-amino acid-containing dipeptides were synthesized using alcalase in ethanol with more than 50% yield (Table 2). Surprisingly, we found that alcalase not only catalyzed the incorporation of D-amino acid esters as nucleophiles but also the incorporation of N-protected D-amino acids as acyl donors.

16.

17.

18. 19.

Acknowledgment We thank Zhongli

Huang for obtaining

the HPLC data. 20.

References 1.

2. 3. 4. 5.

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Zaks, A. and Klibanov, A. M. Enzymatic catalysis in nonaqueous solvents. J. Biol. Chem. 1988, 263, 3194-3201 Klibanov, A. M., Samokhin, K. M., Martinek, K., and Berezin, I. V. A new approach to preparative enzymatic synthesis. Biotechnol. Bioeng. 1977, 19, 1351-1361 Kullmann, W., Enzymatic synthesis of Leu- and Met-enkephalin. Biochem. Biophys. Res. Comm. 1979, 91,693-698 Kimura, Y., Nakanishi, K., and Matsuno, R. Enzymatic synthesis of the precursor of Leu-enkephalin in water-immiscible organic solvent systems. Enzyme Microb. Technol. 1990, 12, 273-280 Nakanishi, K., Takeuchi, A., and Matsuno, R. Long-term continuous synthesis of aspartame precursor in a column reactor with an immobilized thermolysin. Appl. Microbial. Biotechnol. 1990,32,633636 Kimura, Y., Muraya, K., Araki, Y., Matsuoka, H., Nakanishi, K.,

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